The Pseudomonas syringae type III effector AvrRpt2 functions downstream or independently of SA to promote virulence on Arabidopsis thaliana

AvrRpt2, a Pseudomonas syringae type III effector protein, functions from inside plant cells to promote the virulence of P. syringae pv. tomato strain DC3000 (PstDC3000) on Arabidopsis thaliana plants lacking a functional copy of the corresponding RPS2 resistance gene. In this study, we extended our understanding of AvrRpt2 virulence activity by exploring the hypothesis that AvrRpt2 promotes PstDC3000 virulence by suppressing plant defenses. When delivered by PstDC3000, AvrRpt2 suppresses pathogen-related (PR) gene expression during infection, suggesting that AvrRpt2 suppresses defenses mediated by salicylic acid (SA). However, AvrRpt2 promotes PstDC3000 growth on transgenic plants expressing the SA-degrading enzyme NahG, indicating that AvrRpt2 does not promote bacterial virulence by modulating SA levels during infection. AvrRpt2 general virulence activity does not depend on the RPM1 resistance gene, as mutations in RPM1 had no effect on AvrRpt2-induced phenotypes. Transgenic plants expressing AvrRpt2 displayed enhanced susceptibility to PstDC3000 strains defective in type III secretion, indicating that enhanced susceptibility of these plants is not because of suppression of defense responses elicited by other type III effectors. Additionally, avrRpt2 transgenic plants did not exhibit increased susceptibility to Peronospora parasitica and Erysiphe cichoracearum, suggesting that AvrRpt2 virulence activity is specific to P. syringae.     

The Erwinia amylovora avrRpt2EA gene contributes to virulence on pear and AvrRpt2EA is recognized by Arabidopsis RPS2 when expressed in pseudomonas syringae

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In an attempt to identify genes induced during infection of host plants, we identified and cloned a putative effector gene, avrRpt2EA. The deduced amino-acid sequence of the translated AvrRpt2EA protein is homologous to the effector protein AvrRpt2 previously reported in Pseudomonas syringae pv. tomato. These two proteins share 58% identity (70% similarity) in the functional domain; however, the secretion and translocation signal domain varied. The avrRpt2EA promoter region contains a typical 'hrp box,' which suggests that avrRpt2EA is regulated by the alternative sigma factor, HrpL. avrRpt2EA was detected in all E. amylovora strains tested but not in other closely related Erwinia species. An avrRpt2EA deletion mutant was reduced in its ability to cause systemic infection on immature pear fruits as compared with the wild-type strain, indicating that avrRpt2EA acts as a virulence factor on its native host. Growth of P. syringae pv. tomato DC3000 expressing avrRpt2EA was 10-fold higher than that of P. syringae pv. tomato DC3000 in an Arabidopsis rps2 mutant, indicating that avrRpt2EA promotes virulence of P. syringae pv. tomato DC3000 on Arabidopsis similar to P. syringae pv. tomato avrRpt2. When avrRpt2EA was expressed in P. syringae pv. tomato DC3000 in its native form, a weak hypersensitive response (HR) was induced in Arabidopsis; however, a hybrid protein containing the P. syringae pv. tomato avrRpt2 signal sequence, when expressed from the P syringae pv. tomato avrRpt2 promoter, caused a strong HR. Thus, the signal sequence and promoter of avrRpt2EA may affect its expression, secretion, or translocation, singly or in combination, in P. syringae pv. tomato DC3000. These results indicated that avrRpt2EA is genetically recognized by the RPS2 disease resistance gene in Arabidopsis when expressed in P. syringae pv. tomato DC3000. The results also suggested that although distinct pathogens such as E. amylovora and P. syringae may contain similar effector genes, expression and secretion of these effectors can be under specific regulation by the native pathogen.     

Characterization of the Pseudomonas syringae pv. tomato AvrRpt2 protein: demonstration of secretion and processing during bacterial pathogenesis

Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) expressing avrRpt2 is specifically recognized by plant cells expressing RPS2 activity, resulting in localized cell death and plant resistance. Furthermore, transient expression of this bacterial avrRpt2 gene in plant cells results in RPS2-dependent cell death. This indicates that the AvrRpt2 protein is recognized inside RPS2 plant cells and is sufficient for the activation of disease resistance-mediated cell death in planta. We explored the possibility that Pst DC3000 delivers AvrRpt2 protein to plant cells via the hrp (type III) secretion pathway. We now provide direct evidence that mature AvrRpt2 protein is secreted from Pst DC3000 and that secretion is hrp dependent. We also show that AvrRpt2 is N-terminally processed when Arabidopsis thaliana plants are infected with Pst DC3000 expressing avrRpt2. Similar N-terminal processing of AvrRpt2 occurred when avrRpt2 was stably expressed in A. thaliana. No cleavage of AvrRpt2 was detected in bacteria expressing avrRpt2 in culture or in the plant extracellular fluids. The N-terminus of AvrRpt2 was not required for RPS2 recognition in planta. However, this region of AvrRpt2 was essential for Pst DC3000-mediated elicitation of RPS2-dependent cell death in A. thaliana leaves.     

The Pseudomonas syringae type III effector AvrRpt2 promotes virulence independently of RIN4, a predicted virulence target in Arabidopsis thaliana

AvrRpt2, an effector protein from Pseudomonas syringae pv. tomato (Pst), behaves as an avirulence factor that activates resistance in Arabidopsis thaliana lines expressing the resistance gene RPS2. AvrRpt2 can also enhance pathogen fitness by promoting the ability of the bacteria to grow and to cause disease on susceptible lines of A. thaliana that lack functional RPS2. The activation of RPS2 is coupled to the AvrRpt2-induced disappearance of the A. thaliana RIN4 protein. However, the significance of this RIN4 elimination to AvrRpt2 virulence function is unresolved. To clarify our understanding of the contribution of RIN4 disappearance to AvrRpt2 virulence function, we generated new avrRpt2 alleles by random mutagenesis. We show that the ability of six novel AvrRpt2 mutants to induce RIN4 disappearance correlated well with their avirulence activities but not with their virulence activities. Moreover, the virulence activity of wild-type AvrRpt2 was detectable in an A. thaliana line lacking RIN4. Collectively, these results indicate that the virulence activity of AvrRpt2 in A. thaliana is likely to rely on the modification of host susceptibility factors other than, or in addition to, RIN4.     

The Pseudomonas syringae avrRpt2 gene contributes to virulence on tomato

In order to cause disease on plants, gram-negative phytopathogenic bacteria introduce numerous virulence factors into the host cell in order to render host tissue more hospitable for pathogen proliferation. The mode of action of such bacterial virulence factors and their interaction with host defense pathways remain poorly understood. avrRpt2, a gene from Pseudomonas syringae pv. tomato JL1065, has been shown to promote the virulence of heterologous P. syringae strains on Arabidopsis thaliana. However, the contribution of avrRpt2 to the virulence of JL1065 has not been examined previously. We show that a mutant derivative of JL1065 that carries a disruption in avrRpt2 is impaired in its ability to cause disease on tomato (Lycopersicon esculentum), indicating that avrRpt2 also acts as a virulence gene in its native strain on a natural host. The virulence activity of avrRpt2 was detectable on tomato lines that are defective in either ethylene perception or the accumulation of salicylic acid, but could not be detected on a tomato mutant insensitive to jasmonic acid. The enhanced virulence conferred by the expression of avrRpt2 in JL1065 was not associated with the suppression of several defense-related genes induced during the infection of tomato.     

RPS2, an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2.

A molecular genetic approach was used to identify and characterize plant genes that control bacterial disease resistance in Arabidopsis. A screen for mutants with altered resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) expressing the avirulence gene avrRpt2 resulted in the isolation of four susceptible rps (resistance to P. syringae) mutants. The rps mutants lost resistance specifically to bacterial strains expressing avrRpt2 as they retained resistance to Pst strains expressing the avirulence genes avrB or avrRpm1. Genetic analysis indicated that in each of the four rps mutants, susceptibility was due to a single mutation mapping to the same locus on chromosome 4. Identification of a resistance locus with specificity for a single bacterial avirulence gene suggests that this locus, designated RPS2, controls specific recognition of bacteria expressing the avirulence gene avrRpt2. Ecotype Wü-0, a naturally occurring line that is susceptible to Pst strains expressing avrRpt2, appears to lack a functional allele at RPS2, demonstrating that there is natural variation at the RPS2 locus among wild populations of Arabidopsis.     

Mutations in the Pseudomonas syringae avrRpt2 gene that dissociate its virulence and avirulence activities lead to decreased efficiency in AvrRpt2-induced disappearance of RIN4

The avrRpt2 gene from Pseudomonas syringae pv. tomato exhibits avirulence activity on Arabidopsis expressing the resistance gene RPS2 but promotes bacterial virulence on susceptible rps2 Arabidopsis. To understand the functional relationship between the avirulence and virulence activities of avrRpt2, we analyzed a series of six avrRpt2 mutants deficient in eliciting the RPS2-dependent hypersensitive response. We show that the mutants are also severely impaired in triggering RSP2-dependent resistance. Four of these mutants are severely impaired in their virulence activity, whereas two alleles, encoding C-terminal deletions of AvrRpt2, retain significant but slightly reduced virulence activity. Thus, the avirulence and virulence activities of avrRpt2 can be genetically uncoupled. We tested the ability of the two C-terminal deletion mutants to trigger AvrRpt2-induced elimination of the Arabidopsis RIN4 protein and show that they retain this activity but are less efficient than wild-type AvrRpt2. Thus, reduced AvrRpt2 virulence activity is correlated with reduced efficiency in the induction of RIN4 disappearance. This suggests that an alteration in kinetics of RIN4 disappearance triggered by the C-terminal deletion mutants may provide the mechanistic basis for the uncoupling of the avirulence and virulence activities of avrRpt2.     

Mutational analysis of the Arabidopsis RPS2 disease resistance gene and the corresponding pseudomonas syringae avrRpt2 avirulence gene

Plants have evolved a large number of disease resistance genes that encode proteins containing conserved structural motifs that function to recognize pathogen signals and to initiate defense responses. The Arabidopsis RPS2 gene encodes a protein representative of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of plant resistance proteins. RPS2 specifically recognizes Pseudomonas syringae pv. tomato strains expressing the avrRpt2 gene and initiates defense responses to bacteria carrying avrRpt2, including a hypersensitive cell death response (HR). We present an in planta mutagenesis experiment that resulted in the isolation of a series of rps2 and avrRpt2 alleles that disrupt the RPS2-avrRpt2 gene-for-gene interaction. Seven novel avrRpt2 alleles incapable of eliciting an RPS2-dependent HR all encode proteins with lesions in the C-terminal portion of AvrRpt2 previously shown to be sufficient for RPS2 recognition. Ten novel rps2 alleles were characterized with mutations in the NBS and the LRR. Several of these alleles code for point mutations in motifs that are conserved among NBS-LRR resistance genes, including the third LRR, which suggests the importance of these motifs for resistance gene function.     

The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae

Many plant pathogens suppress antimicrobial defenses using virulence factors that modulate endogenous host defenses. The Pseudomonas syringae phytotoxin coronatine (COR) is believed to promote virulence by acting as a jasmonate analog, because COR-insensitive 1 (coil) Arabidopsis thaliana and tomato mutants are impaired in jasmonate signaling and exhibit reduced susceptibility to P. syringae. To further investigate the role of jasmonate signaling in disease development, we analyzed several jasmonate-insensitive A. thaliana mutants for susceptibility to P. syringae pv. tomato strain DC3000 and sensitivity to COR. Jasmonate-insensitive 1 (jin1) mutants exhibit both reduced susceptibility to P. syringae pv. tomato DC3000 and reduced sensitivity to COR, whereas jasmonate-resistant 1 (jar1) plants exhibit wild-type responses to both COR and P. syringae pv. tomato DC3000. A jin1 jar1 double mutant does not exhibit enhanced jasmonate insensitivity, suggesting that JIN1 functions downstream of jasmonic acid-amino acid conjugates synthesized by JAR1. Reduced disease susceptibility in jin1 mutants is correlated with elevated expression of pathogenesis-related 1 (PR-1) and is dependent on accumulation of salicylic acid (SA). We also show that JIN1 is required for normal P. syringae pv. tomato DC3000 symptom development through an SA-independent mechanism. Thus, P. syringae pv. tomato DC3000 appears to utilize COR to manipulate JIN1-dependent jasmonate signaling both to suppress SA-mediated defenses and to promote symptom development.     

Glucocorticoid‐inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death

Pathogenic strains of Pseudomonas syringae pv. tomato carrying the avrRpt2 avirulence gene specifically induce a hypersensitive cell death response in Arabidopsis plants that contain the complementary RPS2 disease resistance gene. Transient expression of avrRpt2 in Arabidopsis plants having the RPS2 gene has been shown to induce hypersensitive cell death. In order to analyze the effects of conditional expression of avrRpt2 in Arabidopsis plants, transgenic lines were constructed that contained the avrRpt2 gene under the control of a tightly regulated, glucocorticoid-inducible promoter. Dexamethasone-induced expression of avrRpt2 in transgenic lines having the RPS2 gene resulted in a specific hypersensitive cell death response that resembled a Pseudomonas syringae-induced hypersensitive response and also induced the expression of a pathogenesis-related gene (PR1). Interestingly, high level expression of avrRpt2 in a mutant rps2–101C background resulted in plant stress and ultimately cell death, suggesting a possible role for avrRpt2 in Pseudomonas syringae virulence. Transgenic RPS2 and rps2 plants that contain the glucocorticoid-inducible avrRpt2 gene will provide a powerful new tool for the genetic, physiological, biochemical, and molecular dissection of an avirulence gene-specified cell death response in both resistant and susceptible plants.     

Two Arabidopsis srfr (suppressor of rps4-RLD) mutants exhibit avrRps4-specific disease resistance independent of RPS4

RPS4 specifies the Arabidopsis disease resistance response to Pseudomonas syringae pv. tomato expressing avrRps4 and was cloned based on the identification of RLD as a naturally occurring susceptible accession. To dissect the molecular and genetic basis of disease resistance, we used a genetic approach to identify suppressor mutations that reactivate the avrRps4-triggered defense response in RLD. In this report, we describe two non-allelic srfr (suppressor of rps4-RLD) mutants, srfr1 and srfr3, that were susceptible to virulent P. syringae pv. tomato strain DC3000, but resistant to DC3000 expressing avrRps4. In quantitative bacterial growth assays, growth of DC3000 was similar in wild-type control and both mutant lines, indicating that basal resistance was not enhanced in srfr1 and srfr3. Growth of DC3000 (avrRps4) was approximately 30-fold lower in srfr1 and srfr3 than in RLD, but intermediate compared with fully resistant Col-0 and transgenic RLD containing RPS4-Col. The srfr1 and srfr3 mutants did not develop spontaneous lesions prior to inoculation or constitutively express the pathogenesis-related gene PR-1. Therefore, srfr1 and srfr3 constitute novel avr-specific mutants that differ from previously described Arabidopsis mutants with elevated disease resistance. The srfr1 and srfr3 mutations were recessive, and both mapped to the bottom of chromosome IV. Genetic analysis indicated that resistance in srfr1 and srfr3 was independent of the rps4-RLD allele, but dependent on a second gene in RLD. We propose that SRFR1 and SRFR3 are negative regulators of avrRps4-triggered gene-for-gene disease resistance.     

Functional analysis of the type III effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the use of a single-copy genomic integration system

Gram-negative phytopathogenic bacteria require a type III secretion apparatus for pathogenesis, presumably to deliver Avr effector proteins directly into plant cells. To extend previous studies of Avr effectors that employed plasmids encoding Avr proteins, we developed a system that permits the integration of any gene into the Pseudomonas syringae genome in single copy. With this system, we confirmed earlier findings showing that P. syringae pv. maculicola strain PsmES4326 expressing the AvrRpt2 effector induces a resistance response in plants with the cognate R gene, RPS2. Chromosomally located avrRpt2, however, provoked a stronger resistance response than that observed with plasmid-expressed AvrRpt2 in RPS2+ plants. Additionally, chromosomal expression of AvrRpt2 conferred a fitness advantage on P. syringae grown in rps2- plants, aiding in growth within leaves and escape to leaf surfaces that was difficult to detect with plasmid-borne avrRpt2. Finally, with the use of the genomic integration system, we found that a chimeric protein composed of the N terminus of the heterologous AvrRpml effector and the C-terminal effector region of AvrRpt2 was delivered to plant cells. Because the C terminus of AvrRpt2 cannot translocate into plant cells on its own, this indicates that the N-terminal region can direct secretion and translocation during an infection, which supports the view that Avr proteins have a modular design. This work establishes a readily manipulatable system to study type III effectors in a biologically realistic context.     

RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens

Colletotrichum higginsianum is a fungal pathogen that infects a wide variety of cruciferous plants, causing important crop losses. We have used map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum . This locus named RCH2 (for recognition of C. higginsianum ) maps in an extensive cluster of disease-resistance loci known as MRC-J in the Arabidopsis ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 ( resistance to Ralstonia solanacearum 1 ) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1-2 , were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum . The screening identified an additional susceptible mutant ( rps4-21 ) that has a 5-bp deletion in the neighboring gene, RPS4-Ws , which is a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 ( Pst - avrRps4 ). The rps4-21 / rrs1-1 double mutant exhibited similar levels of susceptibility to C. higginsianum as the single mutants. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4 . Thus, RPS4-Ws and RRS1-Ws function as a dual resistance gene system that prevents infection by three distinct pathogens.     

A resistance gene product of the nucleotide binding site -- leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo

Resistance (R) genes in plants mediate gene-for-gene disease resistance. The ligand-receptor model, which explains the gene-for-gene specificity, predicts a physical interaction between an elicitor, which is directly or indirectly encoded by an avirulence (avr) gene in the pathogen, and the corresponding R gene product. The nucleotide binding site (NBS) - leucine rich repeats (LRR) class of R genes is the largest known class of R genes. Here we report that an NBS-LRR R protein and its cognate Avr protein form a complex together in the plant cell. The Arabidopsis thaliana R genes RPS2 and RPM1 confer gene-for-gene disease resistance to strains of the phytopathogenic bacterium Pseudomonas syringae carrying the avr genes avrRpt2 and avrB, respectively. Using transient expression of these genes in Arabidopsis leaf mesophyll protoplasts, we first demonstrated that the protoplast system is appropriate for the investigation of the gene-for-gene recognition mechanism. Formation of an in vivo complex containing the RPS2 and AvrRpt2 proteins was demonstrated by co-immunoprecipitation of the proteins following expression of the genes in protoplasts. This complex contained at least one additional plant protein of approximately 75 kDa. Unexpectedly, RPS2 also formed a complex with AvrB. We speculate that complex formation between AvrRpt2 and RPS2 is productive and leads to the elicitation of the resistance response, whilst complex formation between AvrB and RPS2 is unproductive and possibly competes with complex formation between AvrRpt2 and RPS2.     

Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms

A new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1-20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.